Limits...
Biological activity of the thyroid TRK-T3 oncogene requires signalling through Shc.

Roccato E, Miranda C, Ranzi V, Gishizki M, Pierotti MA, Greco A - Br. J. Cancer (2002)

Bottom Line: We demonstrated that the ShcY317F mutant exerts an inhibitory effect on TRK-T3 transforming activity.Such effect can be modulated by the amount of ShcY317F protein and affects the viability of cells expressing TRK-T3 by means of a mechanism involving apoptosis.Our results indicate a definitive role of the adaptor protein Shc in TRK-T3 transforming activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy.

ABSTRACT
The thyroid TRK-T3 oncogene, produced by a chromosomal translocation, is a chimeric, constitutively activated version of the NTRK1/NGF receptor and it is able to transform NIH3T3 cells and differentiate PC12 cells. TRK-T3 oncoprotein triggers multiple signal transduction pathways. Among others, TRK-T3 binds and phosphorylates the Shc and SNT1/FRS2 adaptor proteins both involved in coupling the receptor tyrosine kinase to the mitogen-activated protein kinase pathway by recruiting Grb2/SOS. We were interested in defining the role of Shc in the oncogenesis by TRK-T3. The mutation of TRK-T3 tyrosine 291, docking site for both Shc and FRS2, abrogates the oncogene biological activity. To directly explore the role of Shc we used the ShcY317F mutant, which carries the mutation of a tyrosine residue involved in Grb2 recruitment. We demonstrated that the ShcY317F mutant exerts an inhibitory effect on TRK-T3 transforming activity. Such effect can be modulated by the amount of ShcY317F protein and affects the viability of cells expressing TRK-T3 by means of a mechanism involving apoptosis. Our results indicate a definitive role of the adaptor protein Shc in TRK-T3 transforming activity.

Show MeSH

Related in: MedlinePlus

(A) Effect of ShcY317F on TRK-T3 induced PC12 differentiation. PC12 cells were cotransfected with TRK-T3 and ShcWT (a), ShcY317F (b) or pCGN (c) plasmids, together with VGF8-luc and Renilla plasmids, as described in Materials and Methods and scored for the presence of neurites 3 days later. As control, untreated (d) and NGF-stimulated (50 ng ml−1) (e) PC12 cells are shown. After morphological analysis, transfected PC12 cells were lysates and subjected to immunoblot using anti-TRK and anti-HA antibodies to detect the level of transfected proteins. To quantify the TRK-T3 differentiating activity both luciferase activities were measured using the Dual-Luciferase reported assay system (Promega). The values were expressed relative to Renilla luciferase activity for normalisation. The results presented are an average of three experiments. (B) Microfocus formation assay. One hundred cells from focus NF797 (NIH3T3 cells transformed by TRK-T3 oncogene) transfected with pCGN vector, ShcWT or ShcY317F constructs were combined with 1×105 NIH3T3 cells in 10-cm dishes, and cultured for 2 weeks in medium containing 5% calf serum. The foci were counted after GIEMSA staining. (C) Transforming activity of TRK-T3 in NIH3T3, NWT (NIH3T3 cells stably expressing ShcWT) and NY317F (NIH3T3 cells stably expressing ShcY317F) cells. Transfection and foci selection were performed as described in Materials and Methods. Foci and G418 resistant colonies were either fixed and GIEMSA stained or isolated for further analysis after two weeks of selection. (D) Effect of hygromycin on TRK-T3 foci formation in NWT and NY317F cell lines. Foci selection was performed in 5% serum medium supplemented or not with 400 μg ml−1 of G418 (selectable marker contained in the TRK-T3 expression vector) in the absence or presence of 25 μg ml−1 hygromycin (selectable marker contained in the Shc expression vector). Foci were scored after 2 weeks of selection. For each cell line, the number of foci per μg DNA selected in the absence of hygromycin was set at 100%.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2364237&req=5

fig4: (A) Effect of ShcY317F on TRK-T3 induced PC12 differentiation. PC12 cells were cotransfected with TRK-T3 and ShcWT (a), ShcY317F (b) or pCGN (c) plasmids, together with VGF8-luc and Renilla plasmids, as described in Materials and Methods and scored for the presence of neurites 3 days later. As control, untreated (d) and NGF-stimulated (50 ng ml−1) (e) PC12 cells are shown. After morphological analysis, transfected PC12 cells were lysates and subjected to immunoblot using anti-TRK and anti-HA antibodies to detect the level of transfected proteins. To quantify the TRK-T3 differentiating activity both luciferase activities were measured using the Dual-Luciferase reported assay system (Promega). The values were expressed relative to Renilla luciferase activity for normalisation. The results presented are an average of three experiments. (B) Microfocus formation assay. One hundred cells from focus NF797 (NIH3T3 cells transformed by TRK-T3 oncogene) transfected with pCGN vector, ShcWT or ShcY317F constructs were combined with 1×105 NIH3T3 cells in 10-cm dishes, and cultured for 2 weeks in medium containing 5% calf serum. The foci were counted after GIEMSA staining. (C) Transforming activity of TRK-T3 in NIH3T3, NWT (NIH3T3 cells stably expressing ShcWT) and NY317F (NIH3T3 cells stably expressing ShcY317F) cells. Transfection and foci selection were performed as described in Materials and Methods. Foci and G418 resistant colonies were either fixed and GIEMSA stained or isolated for further analysis after two weeks of selection. (D) Effect of hygromycin on TRK-T3 foci formation in NWT and NY317F cell lines. Foci selection was performed in 5% serum medium supplemented or not with 400 μg ml−1 of G418 (selectable marker contained in the TRK-T3 expression vector) in the absence or presence of 25 μg ml−1 hygromycin (selectable marker contained in the Shc expression vector). Foci were scored after 2 weeks of selection. For each cell line, the number of foci per μg DNA selected in the absence of hygromycin was set at 100%.

Mentions: We first investigated the effect of ShcY317F on TRK-T3 differentiating activity. PC12 cells were cotransfected with TRK-T3 together with HA-tagged ShcWT, ShcY317F or the pCGN empty vector. The results are reported in Figure 4AFigure 4


Biological activity of the thyroid TRK-T3 oncogene requires signalling through Shc.

Roccato E, Miranda C, Ranzi V, Gishizki M, Pierotti MA, Greco A - Br. J. Cancer (2002)

(A) Effect of ShcY317F on TRK-T3 induced PC12 differentiation. PC12 cells were cotransfected with TRK-T3 and ShcWT (a), ShcY317F (b) or pCGN (c) plasmids, together with VGF8-luc and Renilla plasmids, as described in Materials and Methods and scored for the presence of neurites 3 days later. As control, untreated (d) and NGF-stimulated (50 ng ml−1) (e) PC12 cells are shown. After morphological analysis, transfected PC12 cells were lysates and subjected to immunoblot using anti-TRK and anti-HA antibodies to detect the level of transfected proteins. To quantify the TRK-T3 differentiating activity both luciferase activities were measured using the Dual-Luciferase reported assay system (Promega). The values were expressed relative to Renilla luciferase activity for normalisation. The results presented are an average of three experiments. (B) Microfocus formation assay. One hundred cells from focus NF797 (NIH3T3 cells transformed by TRK-T3 oncogene) transfected with pCGN vector, ShcWT or ShcY317F constructs were combined with 1×105 NIH3T3 cells in 10-cm dishes, and cultured for 2 weeks in medium containing 5% calf serum. The foci were counted after GIEMSA staining. (C) Transforming activity of TRK-T3 in NIH3T3, NWT (NIH3T3 cells stably expressing ShcWT) and NY317F (NIH3T3 cells stably expressing ShcY317F) cells. Transfection and foci selection were performed as described in Materials and Methods. Foci and G418 resistant colonies were either fixed and GIEMSA stained or isolated for further analysis after two weeks of selection. (D) Effect of hygromycin on TRK-T3 foci formation in NWT and NY317F cell lines. Foci selection was performed in 5% serum medium supplemented or not with 400 μg ml−1 of G418 (selectable marker contained in the TRK-T3 expression vector) in the absence or presence of 25 μg ml−1 hygromycin (selectable marker contained in the Shc expression vector). Foci were scored after 2 weeks of selection. For each cell line, the number of foci per μg DNA selected in the absence of hygromycin was set at 100%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2364237&req=5

fig4: (A) Effect of ShcY317F on TRK-T3 induced PC12 differentiation. PC12 cells were cotransfected with TRK-T3 and ShcWT (a), ShcY317F (b) or pCGN (c) plasmids, together with VGF8-luc and Renilla plasmids, as described in Materials and Methods and scored for the presence of neurites 3 days later. As control, untreated (d) and NGF-stimulated (50 ng ml−1) (e) PC12 cells are shown. After morphological analysis, transfected PC12 cells were lysates and subjected to immunoblot using anti-TRK and anti-HA antibodies to detect the level of transfected proteins. To quantify the TRK-T3 differentiating activity both luciferase activities were measured using the Dual-Luciferase reported assay system (Promega). The values were expressed relative to Renilla luciferase activity for normalisation. The results presented are an average of three experiments. (B) Microfocus formation assay. One hundred cells from focus NF797 (NIH3T3 cells transformed by TRK-T3 oncogene) transfected with pCGN vector, ShcWT or ShcY317F constructs were combined with 1×105 NIH3T3 cells in 10-cm dishes, and cultured for 2 weeks in medium containing 5% calf serum. The foci were counted after GIEMSA staining. (C) Transforming activity of TRK-T3 in NIH3T3, NWT (NIH3T3 cells stably expressing ShcWT) and NY317F (NIH3T3 cells stably expressing ShcY317F) cells. Transfection and foci selection were performed as described in Materials and Methods. Foci and G418 resistant colonies were either fixed and GIEMSA stained or isolated for further analysis after two weeks of selection. (D) Effect of hygromycin on TRK-T3 foci formation in NWT and NY317F cell lines. Foci selection was performed in 5% serum medium supplemented or not with 400 μg ml−1 of G418 (selectable marker contained in the TRK-T3 expression vector) in the absence or presence of 25 μg ml−1 hygromycin (selectable marker contained in the Shc expression vector). Foci were scored after 2 weeks of selection. For each cell line, the number of foci per μg DNA selected in the absence of hygromycin was set at 100%.
Mentions: We first investigated the effect of ShcY317F on TRK-T3 differentiating activity. PC12 cells were cotransfected with TRK-T3 together with HA-tagged ShcWT, ShcY317F or the pCGN empty vector. The results are reported in Figure 4AFigure 4

Bottom Line: We demonstrated that the ShcY317F mutant exerts an inhibitory effect on TRK-T3 transforming activity.Such effect can be modulated by the amount of ShcY317F protein and affects the viability of cells expressing TRK-T3 by means of a mechanism involving apoptosis.Our results indicate a definitive role of the adaptor protein Shc in TRK-T3 transforming activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milan, Italy.

ABSTRACT
The thyroid TRK-T3 oncogene, produced by a chromosomal translocation, is a chimeric, constitutively activated version of the NTRK1/NGF receptor and it is able to transform NIH3T3 cells and differentiate PC12 cells. TRK-T3 oncoprotein triggers multiple signal transduction pathways. Among others, TRK-T3 binds and phosphorylates the Shc and SNT1/FRS2 adaptor proteins both involved in coupling the receptor tyrosine kinase to the mitogen-activated protein kinase pathway by recruiting Grb2/SOS. We were interested in defining the role of Shc in the oncogenesis by TRK-T3. The mutation of TRK-T3 tyrosine 291, docking site for both Shc and FRS2, abrogates the oncogene biological activity. To directly explore the role of Shc we used the ShcY317F mutant, which carries the mutation of a tyrosine residue involved in Grb2 recruitment. We demonstrated that the ShcY317F mutant exerts an inhibitory effect on TRK-T3 transforming activity. Such effect can be modulated by the amount of ShcY317F protein and affects the viability of cells expressing TRK-T3 by means of a mechanism involving apoptosis. Our results indicate a definitive role of the adaptor protein Shc in TRK-T3 transforming activity.

Show MeSH
Related in: MedlinePlus